PROJECT SUMMARY Regeneration of native skin elements – hair follicles, sweat glands and adipose tissue, is a highly thought after outcome of wound healing. While, in principle, very large skin wounds in adult mice can spontaneously regenerate new hair follicles and new adipocytes, commonly studied small wounds in mice and clinical wounds in humans heal with a far less desirable fibrotic scarring. If and how adult skin wounds can be directed to replace the natural tendency for healing with a scar with regeneration of native skin elements remains unknown. This application is inspired by a serendipitous discovery that adding an antigen to our novel biomaterial, the Microporous Annealed Particle (MAP) hydrogel, can induce regeneration of new hair follicles when added into normally fibrotic small mouse skin wounds. This immunomodulatory MAP gel provides wound-resident immune cells with the molecular triggers that elicit an adaptive immune response to enhance macrophage responses. Further, our studies on naturally regenerating very large skin wound model show that macrophage-fibroblast interactions are essential for stimulating new hair follicle regeneration. Through an integrated bioengineering, bioinformatic and experimental approach, this application will focus on testing our new hypothesis that by engineering MAP gels to have specific immune triggers, interactions between T-cells, macrophages, and fibroblasts in the wound can transform normally profibrotic healing response into highly desirable regenerative response. The first aim of the proposed research is to mechanistically establish the lymphocyte and macrophage subsets and the molecular signaling pathways required for MAP formulations we have created to elicit hair follicle regeneration. This will be achieved using bioinformatic analyses of transcriptomics, proteomic, and functional profiling at single-cell resolution. confirmed with in vivo loss of function/ transgenic mouse studies lacking key immune pathways or cells MAP gels. The second aim is to engineer new types of immunomodulatory MAP gels designed to maximally induce T-cells and macrophage pro-regenerative signals while minimizing pro-fibrotic signals using a high-throughput in vitro assay. The third aim is to determine how MAP gel-induced immune signals enhance lineage plasticity of wound fibroblasts that is prerequisite for new hair regeneration. This will be achieved via an advanced bioinformatic analysis on single-cell transcriptomic data and functional gain- and loss-of-function studies on wound immune cells and fibroblasts. The study premise is based on newly accepted-for-publication and extensive preliminary data. The proposed studies are significant because they will establish new immune cell-driven mechanism for enhancing fibroblast plasticity and activating embryonic-like regeneration of native skin elements in adult wounds. The proposed studies are innovative because they will establish new types of immune-modulating bi...